B族G蛋白偶联受体PAC1可调控表达体系的建立

PAC1是神经肽垂体腺苷酸环化酶激活多肽(Pituitary adenylate cyclase activating polypeptide,PACAP)的特异受体,属于B族G蛋白偶联受体,介导PACAP的神经递质、神经调质、神经保护、抗神经损伤及调控神经再生等功能,PAC1高表达和神经损伤、肿瘤等生理病理过程密切相关。为了深入了解PAC1的功能,构建PAC1可调控表达的细胞系,通过优化的四环素控制表达系统实现PAC1在中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞的强力霉素(doxycycline,Dox)依赖的可控表达。首先通过双酶切将编码PAC1和增强型黄色荧光蛋白(EYFP,enhanced yellow fluorescent protein)的融和基因PAC1-EYFP克隆到pTRE-Tight载体上,获得重组载体pTRE-PAC1-EYFP;基因测序鉴定正确后将新型的四环素调节元件载体pTet-on advanced和反应元件载体pTRE-PAC1-EYFP分别转入CHO细胞中,G418和潮霉素(Hygromycin)双抗筛选阳性克隆PAC1-Tet-CHO,使用梯度浓度四环素类似物强力霉素Dox诱导PAC1-EYFP表达,48 h后检测受体表达水平,并通过MTT法检测不同PAC1表达水平的细胞增殖活性。荧光检测和Western印迹结果显示,成功获得了具有良好诱导性的Dox依赖的PAC1可控表达的细胞系,这些细胞株在传10代后仍能稳定地可控表达PAC1。MTT结果显示PAC1表达水平越高,细胞增殖活性越强。成功所构建的Dox依赖的PAC1可控表达细胞系,为PAC1的生物学功能的深入研究奠定了基础。     

B族G蛋白偶联受体PAC1的N端基序HSDCIF对其二聚化和上膜运输的影响

B族G蛋白偶联受体（G protein-coupled receptors, GPCRs）PAC1是垂体腺苷酸环化酶激活多肽（pituitary adenylate cyclase activating polypeptide, PACAP）的特异受体,介导PACAP神经保护等功能,是神经系统疾病药物开发的重要靶点之一. HSDCIF（His-Ser-Asp-Cys-Ile-Phe）为位于PAC1的N端胞外1区（extracellar domain 1, EC1）的一段短肽序列,与特定负责激活PAC1受体的激动域PACAP（1-6）具有极高的同源性.利用基因敲除技术构建出缺陷HSDCIF基序的PAC1突变体（简称D PAC1）|利用基因工程原理和技术构建系列真核表达重组载体,包括融合了增强型黄色荧光蛋白（enhanced yellow fluorescent protein, EYFP）的表达载体D-PAC1-EYFP|用于生物发光能量转移（bioluminescence resonance energy transfer, BRET）检测的D-PAC1-Rluc|以及用于双分子荧光互补（bimolecular fluorescence complementation, BiFC）实验的D-PAC1-EYFP/N和D-PAC1-EYFP/C.免疫荧光检测（immunofluorescence assay）测定D PAC1的表达|荧光共聚焦显微观察D-PAC1的细胞运输,然后通过 Western印迹、BRET与BiFC方法来检测D PAC1的二聚化情况,综合评价HSDCIF基序对PAC1二聚化和在细胞中定位的影响.检测结果显示,缺陷HSDCIF基序的突变体D PAC1不能发生二聚化,也不能正常的进行上膜运输,而是滞留在内质网中,同时外源化学合成的寡肽HSDCIF可以竞争性地抑制正常PAC1的二聚化.     

启动子荧光素酶报告系统检测低浓度过氧化氢激活神经肽PACAP特异受体PAC1-R启动子的作用

垂体腺苷酸环化酶激活多肽（pituitary adenylate cyclase activating polypeptide,PACAP）特异受体PAC1-R (PACAP receptor 1)是神经系统疾病药物开发的重要靶点。为了研究其表达的生物学机制,本研究克隆了PAC1-R基因转录起始位点上游从-2 500到+26的2 526 bp启动子片段,构建PAC1-R启动子驱动的荧光素酶基因报告载体pGL3-PAC1-Rp,并确证PAC1-R启动子荧光素酶报告系统在小鼠脑神经瘤细胞Neuro-2a和人神经母细胞瘤细胞SH-SY5Y中工作正常。运用此PAC1-R启动子荧光素酶报告系统,首次发现低浓度过氧化氢（hydrogen peroxide,H2O2）有效激活PAC1-R启动子,此作用可被转录因子特化蛋白1（specificity protein 1,SP1）抑制剂光神霉素A（mithramycin A）所抑制,提示SP1参与介导H2O2对PAC1-R启动子的激活作用。生物信息学分析显示,PAC1-R启动子含有多个SP1结合位点。PAC1-R启动子的荧光素酶报告系统的构建为深入探索PAC1-R高表达的作用与机制奠定了基础,低浓度H2O2对PAC1-R启动子激活作用的发现有助于深入诠释低浓度活性氧的生理学作用。     

基于UPLC-Q-Exactive-Orbitrap-MS、网络药理学和实验验证探讨裸花紫珠抗H1N1活性成分及其作用机制

本实验首先对裸花紫珠的提取物进行萃取,得到不同的萃取部位,然后对各部位进行抗甲型流感病毒H1N1型(H1N1)活性筛选,确定裸花紫珠乙酸乙酯萃取部位(EACN)具有较好的体外抗H1N1的活性。采用超高效液相色谱-四级杆-静电场轨道阱联用(UPLC-Q-Exactive-Orbitrap-MS)技术对EACN进行定性分析。基于定性分析的结果,进一步使用数据挖掘和网络药理学,评估药物的活性成分、关键靶点和关键通路,最后使用H1N1感染BALB/c小鼠模型对预测结果进行验证。结果显示,在各个萃取部位中,乙酸乙酯部位体外抗H1N1效果最佳,半数抑制浓度(IC₅₀)为51μg/mL,半数细胞毒浓度(CC₅₀)为859.4μg/mL,选择指数(SI,CC₅₀/IC₅₀)为16.85。从EACN中鉴定出34种化合物,在此基础上,利用网络药理学,筛选出和EACN抗H1N1相关的药物靶点67个,靶点共涉及生物过程491个,与抗H1N1相关的通路147个。H1N1感染BALB/c小鼠实验结果发现,EACN能改善H1N1...     

1株抗流感病毒H1N1红树林内生放线菌的分离与鉴定

采用CPE-MTT方法筛选从海漆叶部分离到的具有抗H1N1病毒活性的内生放线菌,对活性较强的菌株HA12207进行形态学和生理生化特性的研究,并对其16S r DNA序列进行系统发育分析。结果表明,菌株HA12207发酵液稀释20倍后对H1N1病毒的抑制率达到76.5%,HA12207与Tsukamurella tyrosinosolvens IMMIBD-1397T(YI2246)的形态和生理生化特征最为接近,与其16S r DNA序列相似性为99.9%,且在发育树上聚为一个分支。因此将菌株HA12207鉴定为T.tyrosinosolvens,其发酵液具有较强的体外抗H1N1病毒活性,值得进一步研究。     

抗流感病毒H1N1放线菌的筛选及菌株HA10211的鉴定

采用CPE和MTT方法对分离自红树林土壤样品的211株放线菌进行抗H1N1病毒活性筛选,获得28株活性菌株,其中菌株HA10211发酵液稀释20倍时对H1N1病毒的抑制率达到92.2%。菌株HA10211的16S rRNA基因序列与Isoptericola chiayiensis 06182M-1T具有最高同源性(99.3%),在发育树上聚为同一个分支,二者DNA-DNA杂交率为83.2%。依据形态和生理生化特征、系统发育分析和DNA-DNA杂交结果,鉴定菌株HA10211为嘉义白蚁菌(Isoptericola chiayiensis)。     

糖尿病相关VPAC、PAC受体及其药物研发的进展

VPAC、PAC受体是近年发现的开发2型糖尿病药物的重要靶标,垂体腺苷酸环化酶激活多肽(PACAP)、血管活性肠肽(VIP)则是它们的天然配体,与受体结合激活后能促进葡萄糖依赖性胰岛素分泌,既能保护细胞又不引起胰高血糖素分泌和糖原分解,因此其配体相关化合物的开发研究成为开发2型糖尿病药物的重要方向。目前针对VPAC、PAC受体开发的配体类似物主要包括了肽类和非肽类小分子两大类,但它们仍然存在诸多不足,至今仅有一个药物进入到临床研究阶段。本综述系统总结了目前针对VPAC、PAC受体为靶标的肽类和非肽小分子药物的开发研究现状,可为将来此类药物的研发提供参考。     

肝细胞中活化转录因子ATF6抑制SREBP1的转录活性

内质网膜定位的活化转录因子ATF6和SREBP1均是经过蛋白酶切水解激活,激活后的ATF6(N)和SREBP1(N)进入细胞核内,分别指导内质网膜未折叠蛋白聚集反应相关基因和脂肪酸合成相关基因的表达.研究发现,肝细胞内葡萄糖饥饿激活ATF6并抑制SREBP1的转录活性及其靶基因的表达.过表达ATF6(N)能够抑制SREBP1介导的转录及其下游基因的表达.免疫共沉淀实验显示,ATF6(N)在细胞核内结合SREBP1(N),这种结合在无糖状况下增强.不同功能区缺失分析表明,ATF6和SREBP1通过亮氨酸拉链(leucinezipper)功能区相互作用.在葡萄糖饥饿状况下,ATF6对SREBP1转录活性的抑制保证了细胞基本生命活动所需要的能量.     

氮离子束注入燕麦种子对M1-M2代幼苗生理生化指标的影响

采用能量为25keV的不同剂量(4×1016N /cm2、6×1016N /cm2、8×1016N /cm2、12×1016N /cm2)N 束注入燕麦种胚,研究了N 离子束注入后,燕麦M1代和M2代种子的发芽率,幼苗体内过氧化物酶(POD)、过氧化氢酶(CAT)和超氧化物歧化酶(SOD)活性,叶绿素含量、丙二醛(MDA)含量、可溶性糖及可溶性蛋白含量的影响。实验结果表明:以12×1016N /cm2剂量处理燕麦较合适,能提高燕麦M1和M2代种子的发芽率,幼苗POD、CAT活性和可溶性蛋白含量,有利于幼苗生长。     

氮离子束注入对燕麦M1-M2代幼苗耐盐性的影响

采用能量为25keV的不同剂量(4×1016N /cm2、6×1016N /cm2、8×1016N /cm2、12×1016N /cm2)N 注入燕麦种胚,研究N 离子束注入后,盐胁迫对燕麦M1和M2代幼苗体内超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性,脯氨酸含量、丙二醛(MDA)含量、可溶性糖及可溶性蛋白含量的影响。实验结果表明:对M1代,以6×1016N /cm2剂量处理燕麦较合适,能提高燕麦受盐胁迫影响后幼苗SOD活性和可溶性糖含量。而对M2代,4×1016N /cm2处理剂量能降低MDA含量,提高可溶性糖含量,这都有利于盐胁迫后燕麦幼苗的生长。     

荧光互补与光能量共振转移检测B类G蛋白偶联受体PAC1二聚化

G蛋白偶联受体（G—protein couple receptors,GPCRs）是最大的超家族膜受体,其中它的B家族成员垂体腺苷酸环化酶激活肽（PACl）是垂体腺苷酸环化酶激动多肽（PAcAP）的特异受体,介导PAcAP神经保护等功能,是神经系统疾病药物开发的重要靶点之一。二聚化或寡聚化是GPCRs普遍存在的现象,但是目前尚没有关于PACl形成同源二聚体或寡聚体的报道。为了验证PACl也能进行同源二聚化,该文采用生物发光能量转移（bioluminescence resonance energy transfer,BRET）方法进行检测,结果显示不同浓度梯度共转染中国仓鼠卵巢细胞（Chinesehamsterovary,CHO）的PACl一Rluc与PACl一EYFP重组载体,在底物腔肠素h（coelenterazineh）作用下呈现明显的BRET信号。双分子荧光互补（BiFc）检测显示,带有EYFPN端基因标记的PACl与带有EYFPC端基因标记的PACl共转染CHO细胞,能呈现完整的EYFP荧光信号。同时,Westemblot检测也显示,高表达PACl的细胞中可检测到JPACl二聚体的大分子。因此,PACl是能够进行正常同源二聚化的,这个发现将为后续神经损伤药物的开发奠定全新的理论基础,同时也为其他GPCRs同源二聚化的研究起到启发和借鉴作用。     

Pituitary Adenylate Cyclase-activating Polypeptide Type 1 Receptor (PAC1) Gene Is Suppressed by Transglutaminase 2 Activation

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuroprotective factor through the PACAP type 1 receptor, PAC1. In a previous work, we demonstrated that nerve growth factor augmented PAC1 gene expression through the activation of Sp1 via the Ras/MAPK pathway. We also observed that PAC1 expression in Neuro2a cells was transiently suppressed during in vitro ischemic conditions, oxygen-glucose deprivation (OGD). Because endoplasmic reticulum (ER) stress is induced by ischemia, we attempted to clarify how ER stress affects the expression of PAC1. Tunicamycin, which induces ER stress, significantly suppressed PAC1 gene expression, and salubrinal, a selective inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway of ER stress, blocked the suppression. In luciferase reporter assay, we found that two Sp1 sites were involved in suppression of PAC1 gene expression due to tunicamycin or OGD. Immunocytochemical staining demonstrated that OGD-induced transglutaminase 2 (TG2) expression was suppressed by salubrinal or cystamine, a TG activity inhibitor. Further, the OGD-induced accumulation of cross-linked Sp1 in nuclei was suppressed by cystamine or salubrinal. Together with cystamine, R283, TG2-specific inhibitor, and siRNA specific for TG2 also ameliorated OGD-induced attenuation of PAC1 gene expression. These results suggest that Sp1 cross-linking might be crucial in negative regulation of PAC1 gene expression due to TG2 in OGD-induced ER stress.     

Altered Circadian Food Anticipatory Activity Rhythms in PACAP Receptor 1 (PAC1) Deficient Mice

Light signals from intrinsically photosensitive retinal ganglion cells (ipRGCs) entrain the circadian clock and regulate negative masking. Two neurotransmitters, glutamate and Pituitary Adenylate Cyclase Activating Polypeptide (PACAP), found in the ipRGCs transmit light signals to the brain via glutamate receptors and the specific PACAP type 1 (PAC1) receptor. Light entrainment occurs during the twilight zones and has little effect on clock phase during daytime. When nocturnal animals have access to food only for a few hours during the resting phase at daytime, they adapt behavior to the restricted feeding (RF) paradigm and show food anticipatory activity (FAA). A recent study in mice and rats demonstrating that light regulates FAA prompted us to investigate the role of PACAP/PAC1 signaling in the light mediated regulation of FAA. PAC1 receptor knock out (PAC1-/-) and wild type (PAC1+/+) mice placed in running wheels were examined in a full photoperiod (FPP) of 12:12 h light/dark (LD) and a skeleton photoperiod (SPP) 1:11:1:11 h L:DD:L:DD at 300 and 10 lux light intensity. Both PAC1-/- mice and PAC1+/+ littermates entrained to FPP and SPP at both light intensities. However, when placed in RF with access to food for 4–5 h during the subjective day, a significant change in behavior was observed in PAC1-/- mice compared to PAC1+/+ mice. While PAC1-/- mice showed similar FAA as PAC1+/+ animals in FPP at 300 lux, PAC1-/- mice demonstrated an advanced onset of FAA with a nearly 3-fold increase in amplitude compared to PAC1+/+ mice when placed in SPP at 300 lux. The same pattern of FAA was observed at 10 lux during both FPP and SPP. The present study indicates a role of PACAP/PAC1 signaling during light regulated FAA. Most likely, PACAP found in ipRGCs mediating non-image forming light information to the brain is involved.     

Novel stable PACAP analogs with potent activity towards the PAC1 receptor

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38- or 27-amino acid neuropeptide with promising therapeutic applications for the treatment of several pathophysiological states related to neurodegenerative diseases. However, its use for therapeutic applications is actually limited by its restricted bioavailability and rapid degradation. Therefore, metabolically stable PACAP analogs represent promising tools to further investigate the physiological roles of PACAP and ascertain its usefulness in some clinical conditions. In this study, derivatives of PACAP27 and PACAP38 have been rationally designed to develop PAC1 receptor agonists resistant to peptidase action. Results showed that N-terminal modifications confer resistance to dipeptidyl peptidase IV, a major proteolytic process involved in PACAP degradation. Moreover, in vitro incubation of both PACAP isoforms in human plasma revealed that PACAP38 is rapidly metabolized, with a half-life of less than 5 min, while PACAP27 was stable in these experimental conditions. Hence, following the elucidation of its plasmatic metabolites, PACAP38 was modified at its putative endopeptidase and carboxypeptidase sites of cleavage. All peptide analogs were tested for their ability to bind the PAC1 receptor, as well as for their potency to induce calcium mobilization and inhibit PC12 cell proliferation through the PAC1 receptor. This approach revealed two leading compounds, i.e. acetyl-[Ala¹⁵, Ala²⁰]PACAP38-propylamide and acetyl-PACAP27-propylamide, which exhibited improved metabolic stability and potent biological activity. This study describes innovative data related to PACAP metabolism in human plasma and depicts the development of a metabolically stable PACAP38 analog, acetyl-[Ala¹⁵, Ala²⁰]PACAP38-propylamide, which behaves as a super-agonist towards the PAC1 receptor.     

Regulatory mechanism of PAC1 gene expression via Sp1 by nerve growth factor in PC12 cells

In addition to VPAC1 and VPAC2, PAC1 is involved in the pleiotropic action of pituitary adenylate cyclase activating polypeptide (PACAP) in the CNS. A luciferase reporter assay for the human PAC1 gene (-2160/+268) revealed that NGF treatment significantly augments the promoter activity of the PAC1 gene. Moreover, the Sp1 site at -282/-273 was shown to be essential for the NGF-augmented promoter activity of the PAC1 gene. Treatment with U0126, an MEK inhibitor, or Mithramycin A, an Sp1 inhibitor, significantly attenuated promoter activity. These results indicate that activation of Sp1 by the Ras/MAPK pathway might participate in neuron specific expression of the PAC1 gene.     

Expression of PAC1 receptor in rat thymus after irradiation

In the present work, PAC1-R (G-protein-coupled receptor specific for PACAP) was detected on cells in the normal thymus. Immunohistochemically PAC1-R was expressed strongly in stromal cells of the thymic medulla. Positive cells were also observed in the thymus of fetal and old adult rats. After 8 Gy irradiation to 9-week-old rats, PAC1-R expressions in the thymus decreased and almost recovered by day 21. The expression of PAC1-R mRNA was weak in the thymus and decreased further after irradiation. The expression almost recovered by day 28. Hip and hip/hop variants, which were not expressed in the normal thymus, were expressed in the thymus on days 3, 5 and 21 after irradiation. The expressions of IL-6 and IL-10 tended to increase initially after irradiation then decreased. Histologically, the thymic structures were destroyed on day 3 after irradiation and the thymus almost recovered by day 21. Thus PACAP is thought to be one of the important factors for cross-talk between cells involved in thymic regeneration.